T-cells act as the control center for the cellular immune response. T-cell activity can be up or down regulated resulting in the attack or ignoring of an antigen, respectively. The ability to down-regulate or become tolerant to specific antigens is crucial for the preservation of “self” antigens (the molecules that are naïve to our bodies for normal function). On the other hand, non-tolerized T-cells attack and destroy antigens that are foreign to our bodies. Examples of foreign bodies are viruses or defective proteins on the surface of cancer cells.
It is beneficial that T-cells attack foreign antigens to preserve the integrity of our bodies, however, situations can arise where attacking T-cells become a problem. For example, after an organ has been transplanted into a body from another human, or even an animal, there are numerous foreign antigens on the surface of the cells of the foreign tissue. The T-cells do not recognize the difference between a tumor and a beneficial kidney transplant. Thus, the T-cells attack the new kidney as though it is harmful to the body. Attack by the T-cells results in the destruction of the new kidney. This event is referred to as rejection of the transplant.
Rejection of a donated organ from another person or animal can be avoided by ensuring that T-cells do not attack the foreign antigens on the cell surfaces of the new tissue. Physicians accomplish this goal by using immunosuppressive drugs to turn off T-cells so they do not attack the foreign antigens of the donated organ. Thus, the foreign organ is tolerated by the body and is not rejected. Unfortunately, immunosuppressive drugs suppress the entire immune system and other foreign antigens, such as those on virally infected cells and tumor cells, go unchecked by T-cells and can multiply without interference from the T-cells. In fact, immunosuppressive drugs knock out other parts of the immune system, for example, B-cells, resulting in the body being susceptible to attack by bacterial, viral, and fungal organisms. Consequently, immunosuppressive drugs are effective in non-rejection of the organ transplant, but also result in risk of infectious organisms. This problem is addressed by keeping the organ transplant recipient in an environment as clear as possible of infectious agents, but keeping the environment completely infectious agent-free is impossible.
One of the marvels of the immune system is that it is designed to recognize each of the millions of antigens individually. When the immune system is presented with the proper antigenic information, the T-cells will attack only that specific antigen(s). Similarly, T-cells can be tolerized against specific antigens if only those antigens are presented to the immune system in the proper manner. Thus, if only the antigens of the transplant organ are presented to the T-cells for tolerance, the T-cells will leave the transplant alone and still stand guard against other infectious agents.
Central to the regulation of the immune response against any given antigen is the role of the helper T cells. These CD4+ cells interact with an antigen presenting cell (APC) expressing a specific antigenic peptide complexed with MHC Class II antigens. It is generally accepted that two signals are required for effective activation of T lymphocytes. Signal one is provided via interactions with the T cell receptor and its specific antigenic peptide complexed with the MHC protein. The second signal is referred to as the costimulatory signal and it is now believed that a major mediator of signal two is delivered via the T cell surface protein CD28. The CD28 ligands, B7.1 (CD80) and B7.2 (CD86), are primarily found on antigen presenting cells (APCs), but are also sometimes expressed on other non-lymphoid tissues. CD28 engagement also results in an increase in CTLA-4 cell surface expression. CTLA-4 is a critical T cell surface receptor and signaling via CTLA-4 results in the down modulation of the T cell response. CTLA-4 knockout mice have early lethality from a severe lymphoproliferative disorder. Crosslinking experiments with anti-CTLA-4 antibodies in the presence of TCR signaling and CD28 crosslinking show hampered T cell activity. Memory T cells were more sensitive to CTLA-4 mediated inhibition than were nayve cells. CTLA-4 engagement by anti-CTLA-4 antibody generates the antigen or target specific immunoregulatory T cells that effectively suppress unwanted antigen or tissue specific immune response. It has been suggested that T cells constitutively expressing the CD25 marker (CD4+/CD25+ T cells) are critical for ensuring peripheral tolerance against self-reactive T cells.
Dendritic cells are antigen presenting cells (DCs) belonging to a family of professional antigen presenting cells (APCs) that are present in small numbers virtually in all organs. DCs are unique in that they are highly mobile and migrate from peripheral tissues to the lymphoid organs via the blood and/or the lymphatics, a property that is not commonly associated with other APCs. A general property of all subtypes of DCs is that they pass through several stages of maturation during their life span. Immature DCs express low levels of MHC class-II and co-stimulatory molecules, but the surface expression of these molecules dramatically increases upon maturation in response to appropriate antigenic or inflammatory stimuli. A number of stimuli, provided by microbial products and inflammatory chemokines [e.g. tumor necrosis factor-α (TNF-α) and IL-1] can induce migration of DCs, and regulate changes in the expression of chemokine receptors and adhesion proteins on their surface. Immature DCs, in the periphery, are specialized for antigen capture by endocytosis or macropinocytosis, however, once matured, the DCs lose their ability to capture the antigen and become highly efficient antigen presenting cells (APCs). These APCs activate antigen specific naïve T cells in the peripheral lymphoid organs, where the antigen is trapped and both cell types co-localize. In addition to their interaction with T cell receptors (TCRs), DCs can activate T cells through several membrane-bound receptor-ligand interactions and through cytokine production. These interactions can significantly affect not only the magnitude but also the qualitative nature of the T cell response. Another remarkable property of DCs is that they maintain their ability to present antigens encountered in the peripheral tissues even after they have migrated to T cell zones in the lymphoid organs. This allows accumulation and persistence (for over 100 h) of MHC class-I/II-peptide complexes. In contrast, the intracellular sequestration of class-II peptide complexes, and a blockade of peptide loading to MHC molecules in immature DCs can delay the presentation of peptides in the context of MHC. These events are regulated by inflammatory stimuli, which facilitate migration of peptide loaded DCs into the T cell zone in lymph nodes. Stable MHC-peptide complexes expressed on the surface of mature DCs form an immunological synapse with naïve T cells resulting in optimal T cell activation.
Engagement of CTLA-4 can induce regulatory T cells and suppress autoimmune responses: CTLA-4 is a critical T cell surface receptor and signaling via CTLA-4 results in the down modulation of the T cell response. Evidence continues to accumulate that the induction of peripheral T cell tolerance in vivo requires CTLA-4 engagement. Further, recent studies have shown that the CD4+CD25+ Treg cells constitutively express CTLA-4 and suggest that signaling via CTLA-4 is essential for the functioning and maintenance of these cells. High affinity B7.1 interaction, but not B7.2 interaction, with CTLA-4 is important in T cell down-modulation and the Ig-constant like c-domain of B7.1 is important in this high affinity interaction. In addition, interactions between B7.1 and CTLA-4 on activated T cells results in T cells with a regulatory phenotype. The target specific tolerogenic effect of CTLA-4 engagement in an alloantigen system as well as in a murine model of Hashimoto's thyroiditis was demonstrated by the inventors. This tolerance induction by CTLA-4 engagement is mediated by CD4+CD25+ Treg cells and the cytokines (IL-10 and/or TGF-β1) produced by those cells.
The burden of immune-mediated diseases is staggering. In the United States, these conditions result in direct and indirect costs that exceed $100 billion. Autoimmune diseases such as rheumatoid arthritis, type I diabetes and multiple sclerosis together affect approximately 5% of the U.S. population. At least 7% of American children are asthmatic, and more than one in five individuals in the United States are affected by allergies. In addition, immune-mediated graft rejection remains a significant obstacle to the successful transplantation of potentially life-saving organs” (NIAID Publication, Jan. 23, 2001).
Effective intervention against inflammation, autoimmune disease and transplant rejection requires the ability to down regulate the immune response. Traditional clinical approaches to these conditions have relied on the administration of immunosuppressive drugs that result in a global attenuation of the immune response. As evidenced by patients suffering from AIDS, or those receiving immunosuppressive agents to prevent transplant rejection, an individual with a weakened immune response is susceptible to a wide range of opportunistic infectious agents and an increased risk for developing malignancies. These potentially fatal side effects continue to be the limiting factors of current immunosuppressive drugs.
Organ transplants from other humans, or in theory other animals, would be feasible if the T-cells that would normally attack antigens on the cells of the transplanted organ were down-regulated.
Granulocyte-macrophage-colony-stimulating-factor (GM-CSF) has the potential, not only to prevent, but also to suppress experimental autoimmune thyroiditis (EAT). GM-CSF induced EAT suppression in mice was accompanied by an increase in the frequency of CD4+CD25+ regulatory T cells that could suppress mouse thyroglobulin (mTg) specific T cell responses in vitro, but the underlying mechanism of this suppression was not elucidated. GM-CSF can induce DCs with a semi-mature phenotype, an important characteristic of DCs that are known to play a critical role in the induction and maintenance of regulatory T cells. Adoptive transfer of CD4+CD25+ T cells from GM-CSF treated and mTg primed donors into untreated, but mTg primed, recipients resulted in decreased mTg specific T cell responses. Furthermore, lymphocytes obtained from these donors and recipients after adoptive transfer produced significantly higher levels of IL-10 relative to mTg primed, untreated, control mice. Administration of anti-IL-10 receptor (αIL-10R) antibody into GM-CSF treated mice abrogated GM-CSF induced suppression of EAT as indicated by increased mTg specific T cell responses, thyroid lymphocyte infiltration, and follicular destruction. Interestingly, in vivo blockade of IL-10 receptor did not affect GM-CSF induced expansion of CD4+CD25+ T cells. However, IL-10 induced immunosuppression was due to its direct effects on mTg specific effector T cells. Taken together, these results indicated that IL-10, produced by CD4+CD25+ T cells that were likely induced by semi-mature DCs, is essential for disease suppression in GM-CSF treated mice.
Though dendritic cells (DCs) are essential for the induction of an effective immune response against foreign antigens, they can also play a critical role in promoting and maintaining tolerance to self-antigens. Modulation of DC phenotype and maturation status in vitro and in vivo can have a profound effect on T cell activation and differentiation, and skew the immune response. Different subsets of DCs can preferentially influence a Th1 or a Th2 type response. Specifically, injection of CD8a+ DCs triggers the development of Th1 cells whereas CD8a− DCs induce Th2-type responses to soluble antigens. Therefore, targeted expansion of a particular DC subset might be used to shift an immune response from one type to another and thereby prevent autoimmune disease development. In addition, DC maturation can be modulated using different cytokines to induce either regulatory T cells or effector T cells.
Neither CD8a+ nor CD8a− DCs can induce optimal T cell responses when they are immature, but become potent activators of T cells when they are matured. While immature DCs, characterized by expression of low levels of co-stimulatory molecules and pro-inflammatory cytokines, can promote anergy; semi-matured DCs that express significant levels of MHC class II and co-stimulatory molecules, but low levels of pro-inflammatory cytokines compared to mature DCs can induce regulatory T cells. Modulation of functional properties of DCs can be an effective therapeutic approach for autoimmune conditions.
Experimental autoimmune thyroiditis is a well established mouse model for Hashimoto's thyroiditis (HT). Hashimoto's thyroiditis is an organ-specific autoimmune disease characterized by lymphocyte infiltration of the thyroid that eventually leads to follicular destruction. In HT, thyroglobulin specific T cells are generated and they migrate to the thyroid. These cells produce IFN-γ, which induces expression of MHC class II on thyrocytes, and results in further expansion and accumulation of activated mTg specific T cells. The mechanism(s) of thyroid destruction, though not completely understood, appears to involve cytokine production by thyroid infiltrating T cells that can facilitate apoptosis of thyrocytes through caspase activation.
Administration of GM-CSF or Flt3-L, potent dendritic cell growth factors, resulted in suppression or augmentation of EAT respectively. Treatment with GM-CSF induced CD8a− DCs and caused a shift in the immune response against thyroglobulin from a Th1 response to a Th2 response, as seen by increased IL-4 production with a concomitant decrease in IFN-γ production. However, GM-CSF induced suppression of EAT was not associated with mere Th2 skewing but also with a selective expansion of CD4+CD25+ regulatory T cells that could suppress mTg specific responses in vitro. CD4+CD25+ regulatory T cells play a critical role in the suppression of autoimmunnity. Depletion or absence of CD4+CD25+ regulatory T cells has been reported to result in the development of autoimmune disease. Although how regulatory CD4+CD25+ T cells suppress autoimmunity is not fully understood, suppressor cytokines, such as IL-10, have been implicated. In GM-CSF treated mice, there was considerable increase in the levels of IL-10, and neutralization of IL-10 in lymphocyte cultures derived from GM-CSF treated mice restored mTg specific T cell responses. Furthermore, lymphocytes from GM-CSF treated mice, that were depleted of CD4+CD25+ T cells showed enhanced mTg specific proliferation with a concomitant decrease in the levels of IL-10 in vitro, suggesting that these cells were the source of IL-10.
Investigation of a direct role of CD4+CD25+ T cells and IL-10 in GM-CSF induced suppression of EAT showed that adoptive transfer of CD4+CD25+ T cells from GM-CSF treated mice into mTg primed mice can suppress mTg specific proliferation, and cells from recipient mice can produce higher levels of IL-10. Furthermore, in vivo blockade of IL-10 receptor can abrogate GM-CSF induced suppression and restore mTg specific T cell responses resulting in the development of EAT. An increase in DCs with a semi-mature phenotype in GM-CSF treated mice was observed. Since semi-matured DCs can induce regulatory T cells, data suggested a mechanism for the induction of regulatory T cells. Results support arolethat CD4+CD25+ T cells and IL-10 play in GM-CSF induced suppression of EAT.
Autoimmune myasthenia gravis (MG)2 is a T cell-dependent, Ab-mediated, organ-specific autoimmune disease. Autoantibodies targeted to the skeletal muscle acetylcholine receptor (AChR) impair neuromuscular transmission resulting in muscle weakness. Current therapies for MG produce nonspecific immune suppression, must usually be continued lifelong to maintain disease control, and are associated with significant chronic side effects and enhanced risk for infection and malignancy.